The overall goal of the studies proposed is to develop detailed understanding of how the enzymes interacting with hydroperoxides of polyunsaturated fatty acids (PUFA) cause electron redistribution in their free radical intermediates. In eukaryotic cells, hydroperoxides of polyunsaturated fatty acids (PUFA) are formed and rapidly converted to signaling molecules in dynamic complexes of several enzymes. The balance of products resulting from separate PUFA oxidation pathways is a subject critical to current pharmaceutical treatment of inflammatory and cardiovascular disorders. This proposal focuses on the mechanisms of electron rearrangements in two representative enzymes forming, and acting on, PUFA hydroperoxides. The specific enzymes chosen for study include manganese lipoxygenases (MnLO and mutants of iron lipoxygenase), that form PUFA hydroperoxides and an animal allene oxide synthase (AOS), that converts PUFA hydroperoxides to downstream signals. Each of the enzymes is chosen because of novel catalytic features. The specific aims are: (1) To examine, by high frequency electron paramagnetic resonance (hfEPR), activation of MnLO by two of its products, 11S- and 13R-hydroperoxides of linoleic acid. (2) To compare reduction potentials of manganese and iron in lipoxygenases using hfEPR. (3) To examine hfEPR spectroscopy and reduction potential of iron lipoxygenase mutated to include sequences that specify manganese binding. (4) To characterize functions and oxidation kinetics of AOS mutated at native tyrosine sites. (5) To obtain very high frequency EPR (vhfEPR) data on the microenvironment of the AOS tyrosine radical. The sensitivity of high frequency EPR (94 GHz), and simulation of EPR spectra, will facilitate many of these studies.